1. Technical Field
The present disclosure relates to a genome editing system and a method thereof for microorganisms, and in particular, to a genome editing system and a method thereof for chromosomal integration/deletion/replacement in Escherichia coli (E. coli).
2. Description of Related Art
Metabolic engineering plays a crucial role for bio-based production of fuels, chemicals, and materials from renewable biomass, and often involves integration of multiple genes to re-direct metabolic fluxes. Hence, chromosomal integration and/or replacement of large DNA into microorganisms remains an important issue in bioengineering.
The most widely used genome editing tools for chromosomal integration/replacement in Escherichia coli may be the endogenous RecA-dependent homologous recombination system, which, however, is inefficient and requires long (about 1,000 bp) flanking regions homologous to the sequence of the desired insertion site (homology arm). Recombineering requires only a pair of short (40-50 bp) homology arms, but the integration efficiency drops sharply for DNA fragments >1,500 bp, and integration of fragments >2,500 bp using 50 bp homology arms is very difficult.
Recently, an RNA-guided editing system based on CRISPR/Cas9-mediated DNA cleavage was developed for programmable, customizable genome engineering. Compared to the conventional genome editing technique, CRISPR/Cas9 can be used to knockout or insert several genes at the same time, and the genome editing technique of the CRISPR/Cas9 is relatively easier than the conventional genome editing technique, so that convenience of genome editing is increased.
Although ensuing reports have demonstrated gene insertion, deletion, and replacement using the CRISPR/Cas9 system, integration of large DNA into E. coli chromosome remains difficult and inefficiency. More specifically, the integration efficiency drops sharply to 35% once DNA fragments are increased to be longer than 5 kb.
Therefore, there is still room for improvement in the efficiency of genome editing by CRISPR/Cas9 system in bacteria and in the future bioengineering applications thereof.